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PubMed1985

Intermediate filament expression and lifespan potential in human somatic cell hybrids.

Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids.

Cell Division↗
PubMed1990

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane.

The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system, qualitative changes in the expression of intermediate filament proteins.

Animals↗
PubMed1987

Monoclonal antibodies against plant proteins recognise animal intermediate filaments.

Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.

Antibody Specificity↗
PubMed1981

Claims that intermediate filaments contain F-actin are unwarranted.

Examination of sectioned, embedded material showed that almost all intermediate filaments normally present in cultured chick embryo fibroblasts, IMR-33 cells, and BHK-21 cells were eliminated by trypsinization. At the same time atypical filaments of intermediate size were formed by close apposition of thin filaments. Previous observations (Buckley et al., 1978, J. Cell Biol. 78:644-652) on negatively stained and on critical-point-dried whole-cell mounts confused these different filament types and, on the basis of heavy meromyosin binding to the atypical intermediate-sized filaments, we suggested that many normal intermediate filaments contain f-actin. The present results show that this suggestion was unwarranted and we therefore retract it.

Actins↗
PubMed1987

Cell type-specific association between two types of spectrin and two types of intermediate filaments.

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside-out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such associations may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.

Animals↗
PubMed1998

Intermediate filaments and their associated proteins: multiple dynamic personalities.

A fusion of mouse and human genetics has now proven that intermediate filaments form a flexible scaffold essential for structuring cytoplasm in a variety of cell contexts. In some cases, the formation of this scaffold is achieved through a newly identified family of intermediate-filament-associated proteins that form cross-bridges between intermediate filaments and other cytoskeletal elements, including actin and microtubules.

Animals↗
PubMed1984

Expression of the intermediate-filament-associated protein synemin in chicken lens cells.

Synemin, a 230-kilodalton polypeptide component of avian muscle and erythrocyte intermediate filaments, is also found in association with the vimentin filaments of lens tissue. In chicken lens cells, synemin is bound to the core vimentin polymer with the same 180-nm periodicity that it exhibits in erythrocytes. Its solubility properties are characteristic of those of intermediate filaments in general and similar to those of synemin in muscle cells and erythrocytes. Synemin appears at an early stage of lens development and undergoes a dramatic accumulation as the epithelial cells elongate and differentiate into fiber cells. In contrast to synemin in cultured skeletal muscle, lens synemin is not confined to postmitotic, terminally differentiating cells but is present in proliferative cells as well. It is lost from the fibers near the center of the lens, as are many other cellular structures including intermediate filaments. These findings provide new information about the occurrence and expression of avian synemin and new insight regarding its presumptive role as a modulator of intermediate-filament function.

Animals↗
PubMed1993

Chicken filensin: a lens fiber cell protein that exhibits sequence similarity to intermediate filament proteins.

Filensin, a 100 kDa, membrane-associated, cytoskeletal protein, is uniquely expressed in the lens fiber cell (Merdes, A., Brunkener, M., Horstmann, H., and Georgatos, S. D. (1991) J. Cell Biol. 115, 397-410). I cloned and sequenced a full-length chicken lens cDNA encoding filensin, also known as CP95 (Ireland, M. and Maisel, H. (1989) Lens and Eye Toxicity Research 6, 623-638). The deduced amino acid sequence of 657 residues contained an internal 280 residue heptad repeat domain with sequence similarities to the rod domain of intermediate filament proteins. The putative filensin rod domain could be divided into three alpha-helical segments (1A, 1B and 2) separated by short, non-helical linkers. The sequence of the amino-terminal end of the filensin rod domain contained the highly conserved intermediate filament segment 1A motif (Conway, J. F. and Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10, 79-98). Allowing conservative amino acid substitutions, the sequence of the carboxy-terminal end of the filensin rod domain was similar to that of the highly conserved intermediate filament rod carboxy terminus. The alpha-helical segments of the shorter filensin rod domain aligned with the corresponding segments of intermediate filament proteins by allowing a gap of four heptad repeats in the amino-terminal half of filensin segment 2. Filensin rod segment 2 contained the characteristic stutter in heptad repeat phasing, nine heptads from the end of the intermediate filament rod. The overall sequence identity between the rod domains of filensin and individual intermediate filament proteins was 20 to 25%, approximately the level of sequence identity observed between intermediate filament proteins of different types. The open reading frame of chicken filensin predicted a 657 amino acid protein with molecular mass of 76 kDa. Embryonic chicken filensin migrated in SDS-PAGE as a triplet of 102, 105 and 109 kDa, while rooster filensin migrated as a 105 and 109 kDa doublet. Antibodies to filensin labeled lens fiber cells but not lens epithelial cells. By immunofluorescence methods filensin was localized to the fiber cell plasma membranes, including the ends of elongated fiber cells.

Amino Acid Sequence↗
PubMed1992

Attachment of steroidogenic lipid droplets to intermediate filaments in adrenal cells.

Light microscopy of living and extracted adrenal cells (Y-1 mouse adrenal tumour cells and cultured bovine fasciculata cells), using Nomarski optics and fluorescence with nile red to stain lipid, revealed in both cell types that lipid droplets remain attached to intermediate filaments when the cells are extracted to prepare these structures. Electron microscopy of thin sections shows the presence of lipid droplets in both cell types. The droplets differ in appearance but are, in both cases, surrounded by a complete capsule 5 nm wide. The droplets in Y-1 cells include those associated with lysosomes and crystalline structures in addition to typical rounded forms. Only the latter type is seen in bovine fasciculata. Intermediate filaments apparently ending in droplets can also be seen. Immunoelectron microscopy with anti-vimentin and Protein A conjugated to gold particles together with measurement of the diameter of these structures identifies them as intermediate filaments. When adrenal cells are permeabilised and extracted under mild or severe conditions using Triton X-100, thin sections showed that lipid droplets remain associated with the cytoskeleton and in particular intermediate filaments. Extraction under mild and severe conditions cleared the cell contents, revealing attachment of intermediate filaments to lipid droplets with greater clarity than in unextracted cells, i.e. homogenised cells or cells subjected to lysis. Such attachment was unequivocally demonstrated in stereo pairs. These observations support our earlier studies showing attachment of droplets to intermediate filaments, which suggests a role for these filaments in intracellular transport of cholesterol.

Adrenal Glands↗
PubMed1985

Keratin type intermediate filaments in sweat gland myoepithelial cells.

A study was undertaken to clarify the origin of sweat gland myoepithelial cells using monoclonal antibodies EKH1, EKH4, and AN3. EKH1 recognizes all classes of intermediate filaments. EKH4 and AN3 recognize keratin type intermediate filaments. Since within the skin, only epithelial cells of ectodermal origin contain keratin, EKH4 and AN3 could be used as ectodermal markers within the skin. Sweat gland myoepithelial cells were labeled by all three antibodies. In contrast, arrector pili muscle and vascular smooth muscle were recognized only by EKH1, but not by EKH4 and AN3. This study demonstrated that myoepithelial cells of sweat glands contain keratin type intermediate filaments and suggested their ectodermal origin. On the other hand, arrector pili muscle and vascular smooth muscle did not contain keratin type intermediate filaments, despite their ultrastructural similarity to myoepithelial cells. Electron microscopic studies using human fetal and adult skin revealed that myoepithelial cells are developed from basal cells of the coiled tip of fetal gland and not from mesenchymal cells. In order to determine the time of appearance of myoepithelial cells during fetal development, embryonic and newborn mouse skin was also examined. It was found that sweat gland myoepithelial cells first appear around 20 weeks of gestation in humans and after birth in mice.

Animals↗
PubMed1991

2,5-Hexanedione exposure alters the rat Sertoli cell cytoskeleton. II. Intermediate filaments and actin.

The effects of 2,5-hexanedione (2,5-HD) exposure on the expression of intermediate filament and actin proteins in adult rat testis was examined during progressive stages of testicular injury. The distribution of vimentin within testis cross sections was examined using immunohistochemistry while rhodamine-labeled phalloidin was used to localize filamentous actin. Keratin was not detected in immunoblots of intermediate filament proteins tested with an antikeratin antibody to investigate the possible reexpression of the prepubertal intermediate filament protein keratin in 2,5-HD-exposed adult testes. However, 2,5-HD exposure did result in a progressively abnormal distribution of actin and vimentin within the seminiferous epithelium with the appearance of a high-molecular-weight protein which was vimentin immunoreactive and not present in control testes.

Actins↗
PubMed1991

Immunohistochemical distribution of simple-epithelial-type keratins and other intermediate filament proteins in the developing human pituitary gland.

An immunohistochemical study of the production of the intermediate filaments [vimentin, cytokeratin, and glial filament acidic protein (GFAP)] during development of the pituitary gland was made by use of fetal and adult human pituitary tissue. Among these intermediate filament proteins in the anterior and intermediate lobes of the pituitary, cytokeratin is the first to appear, followed by GFAP and vimentin. However, only cytokeratin is seen during the period of morphogenesis of the pituitary gland, with the type-II subfamily cytokeratin 8 being the earliest to appear. Among the simple-epithelial-type cytokeratins, cytokeratins 8 and 19 were observed within the pituitary primordium during morphogenesis. Cells immunoreactive for cytokeratins 8 and 19 showed a heterogeneous three-dimensional distribution pattern in Rathke's pouch. Both cytokeratins 8 and 19 tended to be strongly positive at sites in the pituitary primordium where cells had become more loosely arranged (i.e., areas far from the diencephalon) but were only weakly positive in areas in which the epithelial cells were densely packed (i.e., areas closely associated with the diencephalon). It is concluded that, during the period of morphogenesis, Rathke's pouch has the intermediate filaments characteristic of simple epithelium and shows different immunoreactivity for simple-epithelial-type cytokeratins from place to place according to the extent of cellular differentiation.

Cell Differentiation↗
PubMed2001

Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.

Animals↗
PubMed1992

Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments.

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross-linked with the lysine specific cross-linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross-links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15-20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino-terminal helices 1A, 1B and 2A. Both EGS cross-links identified in the rod were also isolated from cross-linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross-linked filaments yielded a third EGS cross-link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy-terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross-links on current filament models are discussed.

Amino Acid Sequence↗
PubMed1987

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis.

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.

Animals↗
PubMed1994

Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type.

Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.

Animals↗
PubMed1985

[Relative spacing of the microtubules and intermediate filaments during fibroblast spreading in culture].

Using double immunofluorescence staining, microtubules and intermediate filaments were shown to have similar distribution in the substrate-spread mouse embryo fibroblasts. However, in cells spreading on the substrate, microtubules grow from the centre to the periphery regions quicker than intermediate filaments. We suppose that microtubules may guide the spreading of intermediate filaments from the perinuclear regions to the periphery of the cell during its spreading.

Animals↗
PubMed1984

Monoclonal antibody to intermediate filament proteins in astrocytes.

A monoclonal antibody was developed using rat astrocytes purified in vitro as the starting antigenic material. Selection of the monoclonal was on the basis of astrocyte binding specificity in brain sections using indirect immunofluorescence techniques. The antibody (RBA2) that was chosen was specific for astrocytes in that it did not stain neurons or oligodendrocytes in frozen brain sections. It did, however, show binding to vascular smooth muscle and meningeal cells. The antigenic determinant(s) was determined to be on filaments of the intermediate-size class in cultured astrocytes and fibroblasts. From analysis of binding patterns in various tissues and in immunoblots, it was found that RBA2 cross-reacted strongly with glial fibrillary acidic protein (GFAP) and desmin. There was a weaker cross-reactivity to a vimentin-associated component. It is proposed that this antibody can be used as an astrocyte and blood vessel marker in brain sections, a vimentin marker in cultures and as a probe of intermediate filament composition and distribution.

Animals↗